Rational design of ICD-inducing nanoparticles for cancer immunotherapy

Nanoparticle-based cancer immunotherapy has shown promising therapeutic potential in clinical settings. However, current research mainly uses nanoparticles as delivery vehicles but overlooks their potential to directly modulate immune responses. Inspired by the endogenous endoplasmic reticulum (ER) stress caused by unfolded/misfolded proteins, we present a rationally designed immunogenic cell death (ICD) inducer named NanoICD, which is a nanoparticle engineered for ER targeting and retention. By carefully controlling surface composition and properties, we have obtained NanoICD that can effectively accumulate in the ER, induce ER stress, and activate ICD-associated immune responses. In addition, NanoICD is generally applicable to various proteins and enzymes to further enhance the immunomodulatory capacity, exemplified by encapsulating catalase (CAT) to obtain NanoICD/CAT, effectively alleviated immunosuppressive tumor microenvironment and induced robust antitumor immune responses in 4T1-bearing mice. This work demonstrates engineered nanostructures’ potential to autonomously regulate biological processes and provides insights into the development of advanced nanomedicines for cancer treatment.

Investigation of the mass of NanoICD bound to ER The mass of NanoICD bound to ER were measured using a QCM-D.Briefly, the Au sensor chips were first activated by immersing them in a solution of deionized water: ammonia water: 30% hydrogen peroxide = 5 : 1 : 1 (v/v/v) in an ice-water bath for 10 minutes.Following activation, the chips were rinsed with deionized water and dried using nitrogen gas.Next, the chips were modified with nanoparticles by immersing them in solutions of nBSA, NanoICD/BSA-20, NanoICD/BSA-40, nCAT, or NanoICD/CAT (1.5 μM) for 24 hours at room temperature in the presence of 2imidothiolane hydrochloride (Traut's Reagent, 24 μM) and tris(2-carboxyethyl)phosphate hydrochloride (TCEP,48 μM).Subsequently, the chips were rinsed with deionized water, dried with nitrogen gas, and put into the standard flow module.Each sensor chip was washed with PBS buffer for 1 h at a 10 μL/min flow rate, and then equilibrated at 2 μL/min until the baseline was stable.Then, freshly extracted ER (according to the manufacturer's instructions) in the flow buffer was injected for 30 min at 2 μL/min.After the binding reached equilibrium, the flow phase was replaced with PBS (flow rate 2 μL/min) to simulate the retro-translocation process of the ER.All of the QCM experiments in this study were operated at 37 °C.

Distribution of intracellular high mobility group box 1 (HMGB-1)
Immunofluorescence imaging was used to study the distribution of intracellular HMGB-1 after different treatments.Briefly, B16F10 cells were seeded in 35 mm confocal dish (Ф =15 mm) at a density of 2 × 10 4 cells/well for a day prior exposure to PBS, PTX (15 μM), ETL (100 μM), nBSA (1.5 μM), and NanoICD/BSA (1.5 μM).After incubation for 12 h, the cells were rinsed with cold PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and permeabilized with 0.1 % Triton X-100 for 10 min.Nonspecific binding sites were blocked by pre-incubation with 5 % FBS in PBS for 30 min, followed by incubation with the primary antibody for 1 h, and then incubated with the Alexa594-conjugated monoclonal secondary antibody for 30 minutes after three washes with PBS.Finally, the cells were stained with DAPI for CLSM analysis.The extracellular content of HMGB-1 was evaluated by an HMGB-1 ELISA kit according to the manufacturer's instructions.

Secretion of ATP
The ATP secretion levels of the cells after different treatments were measured using a commercially available ATP assay kit.Briefly, B16F10 cells were seeded in 12-well plates at a density of 1 × 10 5 cells/well for a day prior exposure to PBS, PTX (15 μM), ETL (100 μM), nBSA (1.5 μM), and NanoICD/BSA (1.5 μM).After incubation for 12 h, the supernatant of the cell culture was collected, and the ATP content was measured using an ATP assay kit following the manufacturer's instructions.
Analysis of the signal pathway that induce ICD Flow cytometric measurement and western blot-based analysis were employed to investigate the signal pathway that NanoICD/BSA induces ICD.For flow cytometry, B16F10 cells were seeded in 12-well plates at a density of 1 × 10 5 cells/well for a day prior exposure to PBS, PTX (15 μM), ETL (100 μM), nBSA (1.5 μM), and NanoICD/BSA (1.5 μM).After incubation for 12 h, the cells were rinsed with cold PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and permeabilized with 0.1 % Triton X-100 for 10 min.Nonspecific binding sites were blocked by pre-incubation with 5 % FBS in PBS for 30 min, followed by incubation with the primary antibody for 1 h, and then incubated with the Alexa488-conjugated monoclonal secondary antibody for 30 minutes after three washes with PBS.Finally, the cells were stained with DAPI for CLSM analysis.For western blot-based analysis, B16F10 cells were seeded in 6-well plates at a density of 2 × 10 5 cells/well overnight and then treated with PBS, PTX (15 μM), ETL (100 μM), nBSA (1.5 μM), and NanoICD/BSA (1.5 μM) for 24 h.After incubation, cells were rinsed with PBS and solubilized in 1% Nonidet P-40 lysis buffer.Homogenates were clarified by centrifugation at 20000g for 15 min at 4 ˚C, and protein concentrations were determined with a BCA assay.Total protein lysates were separated by SDS-PAGE on 10 % SDS acrylamide gels, which were then transferred to PVDF membranes (Millipore, USA).The membranes were incubated with primary antibodies against EIF2α, pEIF2α, and β-Actin (1:1000 dilution) overnight, followed by incubating with an HRP-conjugated secondary antibody (1:2000 dilution) for 1 h.

Anti-metastasis assays
The ability of NanoICD/BSA to prevent tumor metastasis was evaluated using a vaccine assay.Briefly, 1 × 10 6 B16F10 cells were first incubated with ETL (100 μM), PTX (15 μM), nBSA (1.5 μM), and NanoICD/BSA (1.5 μM) for 24 h, then washed and resuspended in PBS.Subsequently, the treated cells and PBS (no vaccination control) were inoculated subcutaneously into the lower flank of 6-week-old female C57BL/6 mice (vaccination).One week later, the mice were intravenously injected with 1 × 10 5 untreated B16F10 cells.The mice were sacrificed on 10 days post-treatment, the lung was collected to evaluate the anti-metastasis ability of NanoICD/BSA.The ability of NanoICD/CAT-PCA to prevent tumor recurrence was evaluated with a similar vaccine assay using a different type of cancer cells.Briefly, 1 × 10 6 4T1 cells were inoculated subcutaneously into the left mammary fat pad of 6-week-old female BALB/c mice.One week later, 200 μL of PBS, nCAT-PCA (4 μM, 10 mg/kg), NanoICD/BSA-PCA (4 μM, 2.66 mg/kg), and NanoICD/CAT-PCA (4 μM, 10 mg/kg) were injected intravenously every 2 days for a total of 3 doses.Subsequently, the mice were intravenously injected with 1 × 10 5 untreated B16F10 cells.The mice were sacrificed on 10 days post-treatment, the lung was collected to evaluate the antimetastasis ability of NanoICD/CAT-PCA.

Flow cytometry analysis
Freshly harvested TDLNs, spleen, and tumor tissues were minced and homogenized using a GentleMACs Dissociator, and then passed through a 70 × 10 −6 m cell strainer to obtain single-cell suspensions.The collected cells were diluted to 1×10 7 cells/mL, and 100 μL of cells were stained with a cocktail of fluorescently conjugated antibodies.For intracellular staining, cells were first permeabilized with 100 μL fixation/permeabilization buffer before adding the antibody cocktail.After staining, the cells were fixed with 4 % paraformaldehyde and analyzed using a flow cytometer.

Fig. S4 .
Fig. S4.Size distribution and TEM images of NanoICD/BSA.The scale bar is 50 nm.

Fig. S10 .
Fig. S10.Difference in gene expression [log2(fold change)] between NanoICD and nBSA-treated B16F10 cells.Genes with significantly high expression are shown in red (P-adj < 0.05), while genes with significantly low expression (P-adj < 0.05) are shown in blue.Gray denotes genes that do not exhibit significantly differences.

Fig. S15 .
Fig. S15.Representative flow cytometry plots display the cross presentation of B16F10-OVA after being treated with PBS, PTX, ETL, nBSA, and NanoICD/BSA (a) and the corresponding quantitative results (b).Data are presented as mean ± s.d.from three biological replicates (n = 3).

Fig. S16 .
Fig. S16.Representative flow cytometry plots display the maturation of BMDC after being stimulated by PBS, PTX, ETL, nBSA, and NanoICD/BSA pre-treated B16F10 cells (a) and the corresponding quantitative results (b).Data are presented as mean ± s.d.from three biological replicates (n = 3).

Fig. S23 .
Fig. S23.Relative enzymatic activities of nCAT and NanoICD/CAT to native CAT.Data are presented as mean ± s.d.from three biological replicates (n = 3).

Fig. S24 .
Fig. S24.Zeta potentials of NanoICD/CAT and under different conditions.Data are presented as mean ± s.d.from three biological replicates (n = 3).

Fig. S25 .
Fig. S25.Flow cytometric analysis of the 4T1 cells after incubated with NanoICD/CAT and NanoICD/CAT-PCA under different conditions.

Fig. S26 .
Fig. S26.CLSM images display the exposure of CRT on cell surface after treated with NanoICD/CAT and NanoICD/CAT-PCA under different conditions.The scale bars are 50 μm.

Fig. S27 .
Fig. S27.The levels of ATP in cellular supernatants after different treatment.Data are presented as mean ± s.d.from three biological replicates (n = 3).

Fig. S28 .
Fig. S28.Individual tumor growth kinetics in different groups.Growth curves were stopped when the first mouse of the corresponding group died.

Fig. S29 .
Fig. S29.H&E staining analysis of major organs from the mice in each treatment group.The scale bar is 200 µm.

Fig. S39 .
Fig. S39.The ICD-inducing activity of NanoICD/BSA in various human cell lines.a, Preapoptotic CRT exposure on cell surface (U87MG and SK-Br-3) after treatment with PBS and NanoICD/BSA.b, ATP levels in cellular supernatants (U87MG and SK-Br-3) after treatment with PBS and NanoICD/BSA.c, Extracellular concentrations of HMGB-1 after treating the cells (U87MG and SK-Br-3) with PBS and NanoICD/BSA.Data are presented as mean ± s.d.from three biological replicates (n = 3).

Table S3 .
Characterization of different NanoICD and the pro-apoptotic CRT exposure on cell surface (PI -CRT + cells) after treatment